Tissue engineering uses a combination of cells, engineering materials and suitable biochemical and physio-chemical factors to improve or replace biological functions. One approach to tissue engineering is to create an environment using cells within an artificially-created support system that attempts to mimic the environment of tissue development in vivo (i.e., like the conditions in the body). Two important components of creating tissue are a scaffold and a bioreactor. A scaffold is a three dimensional (“3D”) structure optimized for cell attachment and tissue growth. The scaffold is inserted into a bioreactor, which is the culture vessel that provides conditions that are dynamic and similar to in vivo conditions.
To achieve the goal of tissue reconstruction, scaffolds must meet specific requirements. A high porosity and an adequate pore size are necessary to facilitate cell seeding and diffusion throughout the whole structure of both cells and nutrients. Biodegradability is often an essential factor since scaffolds should preferably be absorbed by the surrounding tissues without the necessity of a surgical removal. The scaffold is designed so that the rate at which degradation occurs closely coincides with the rate of tissue formation. While the cells are fabricating their own natural matrix structure around themselves, the scaffold provides structural integrity within the body. When the scaffold degrades, the newly formed tissue takes over the mechanical load. For clinical uses, it is desirable for the scaffold material to be injectable.
Cells are often implanted or “seeded” into the scaffolds, which can serve one or more functions: (1) allow cell attachment and migration; (2) deliver and retain cells and biochemical factors; (3) enable diffusion of vital cell nutrients and expressed products; and (4) exert certain mechanical and biological influences to modify the behavior of the cell phase. In general, bioreactors are used to: (1) establish uniform cell distribution on the scaffold; (2) maintain desired amounts of nutrients and gases; and (3) expose developing tissue to physical stimuli, which modifies tissue development.
Many different materials (natural and synthetic, biodegradable and permanent) have been investigated for use as scaffold materials. Most of these materials have been known in the medical field for uses other than tissue engineering, such as bioresorbable sutures. Examples of these materials are collagen and some polyesters. Scaffolds may also be constructed from natural materials. In particular, different derivatives of the extracellular matrix have been studied to evaluate their ability to support cell growth. Proteic materials, such as collagen or fibrin, and polysaccharidic materials, like chitosan or glycosaminoglycans (GAGs), have all proved suitable in terms of cell compatibility, but some issues with potential immunogenicity still remains. Among GAGs hyaluronic acid, possibly in combination with cross linking agents (e.g. glutaraldehyde, water soluble carbodiimide, etc.), is one of the possible choices as scaffold material.
Hyaluronic acid (HA) (also called hyaluronic acid or hyaluronate) is a naturally occurring polymer found in every tissue of the body. It is particularly concentrated in the skin (almost 50% of the HA in the body is found in the skin) and synovial fluid. HA is an anionic, nonsulfated glycosaminoglycan consisting of repeating disaccharides of alternating D-glucuronic acid and N-acetylglucosamine molecules. It is a straight-chained polymer with a molecular weight that varies between 50,000 and 13,000,000 daltons. Hyaluronic acid is naturally present in the pericellular gels, in the fundamental substance of connective tissue and in vertebrate organisms, of which it is one of the chief components, in the synovial fluid of joints, in the vitreous humor, in the human umbilical cord tissues and in rooster combs. One of the chief components of the extracellular matrix, hyaluronan contributes significantly to cell proliferation and migration, and may also be involved in the progression of some malignant tumors.
Hyaluronic acid plays a vital role in many biological processes. For example, hyaluronic acid is applied in the tissue repair process, especially in the early stages of granulation, stabilizing the coagulation matrix and controlling its degradation. When skin is exposed to excessive UVB rays, it becomes inflamed (sunburn) and the cells in the dermis stop producing as much hyaluronan, and increase the rate of its degradation. Hyaluronan degradation products also accumulate in the skin after UV exposure. The application of hyaluronic acid solutions has been found to accelerate healing in patients suffering from sores, wounds and bums. It is also known that hyaluronic acid fractions can be used to facilitate tissue repair, as substitutes for the intraocular fluid, or they can be administered by the intra-articular route to treat joint pathologies.
The physiological activity of HA polymers and oligomers makes it a promising material for a variety of applications. HA gels are popular for cell culture scaffolds in tissue engineering. However, HA must be crosslinked to achieve the proper mechanical properties and not be digested by HA aces enzymes (i.e., angiotensin-converting enzyme). Most commercially available HA hydrogels are chemically crosslinked where the chemicals are cytotoxic. In addition, these chemicals are expensive and drive up the prices of the gels so they are not commercially viable.
Accordingly, there is a need for less expensive HA-hydrogel scaffolds that are non-toxic and have biochemical functionality and improved mechanical stability against degradation.